This project will examine the actions of steroid anesthetics on transmitter-gated membrane channels. The goal of the experiments is to define the mechanisms by which steroids act on a major target, the gamma-amino butyric acid type A (GABA-A) receptor, and determine the site(s) at which steroids bind. Defined combinations of subunits will be examined for their effects on a single channel activity and on responses of populations of channels, to provide insight into the number of sites and mechanisms of action required to explain the actions of steroid anesthetics. Complementary work will use molecular manipulations of the receptor structure, to define the regions of the receptor which are required for recognition of anesthetics and for transducing the effects of anesthetic binding. Stable clonal cell lines expressing high levels of epitope-tagged GABA-A receptors will be generated for biophysical and biochemical studies. The studies of GABA-A receptors will be extended by studies of the actions of steroid anesthetics on glycinergic receptors. Finally, the actions of the intravenous anesthetic pentobarbitol will be compared to those of the steroid anesthetics. The proposed work builds on previous studies in this Program, and is expected to lead to insights into the mechanisms by which steroid anesthetics act at one molecular target, the GABA-A receptor. In conjunction with other projects in this Program, the results will reveal the number(s) of distinct sites and actions of steroids. Furthermore, comparisons with other data obtained in the project and the Program will indicate the range of effects which steroid anesthetics have on several identified membrane channels involved in synaptic transmission. All this information will provide insights into the cellular actions associated with anesthesia.